Jeff:LabNotes/Microbiome/2012-8-23: Difference between revisions

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(Created page with "== ''Tagmentation test using Genomic DNA''' == *Test 4 samples, each with 25 ng genomic DNA starting template *Sample 1: PC in water *Sample 2: DNA is EtOH precipitated and e...")
 
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[[File: 11-13-12 pcr.jpg|400px]]  [[File: 11-13-12 gel1.jpg|400px]]
[[File: 11-14-12 pcr.jpg|400px]]  [[File: 11-14-12 gel1.jpg|400px]]

Revision as of 21:11, 14 November 2012

Tagmentation test using Genomic DNA'

  • Test 4 samples, each with 25 ng genomic DNA starting template
  • Sample 1: PC in water
  • Sample 2: DNA is EtOH precipitated and eluted in water
  • Sample 3: DNA in New prep below w/o Pol I. ALS and NS premixed as to be neutral
  • Sample 4: Pol I treated DNA using New prep

Pol 1 treatment

  • Start with 1 uL DNA
  • Add 1.5 uL ALS, 3min
  • Put on ice and add 1.5 uL NS

New Prep

Template  4 uL    
10x buffer 4 .7 uL    
dNTP .35 uL          
N6(unmodified) .35 uL  
water .4 uL         
Pol I .5 uL         
----------------------------------
Total 7 uL         


  • 37 C for 30 min, 65C for 10 min

Tagmentation

DNA in water  7 uL
HMW buffer  2 uL, (.6 uL for New prep samples for a total of 2 uL)
water (for new prep only) 1.4 uL
1:50 diluted enzyme  1 uL
----------------------
Total 10 uL
  • 55C for 5 min

Protease treatment

  • Add 1 uL 1:100 diluted protease
  • 10 min 50C, 20 min 70C

Klenow

  • Add 1 uL exo- klenow and 1 uL dNTP to each
  • 37C for 15 min, 75 for 20 min

1st PCR round

DNA  13 uL
KAPA Robust mix  15 uL
Orange  .6 uL
blue   .6 uL
50x SYBR .24 uL
--------------------------
Total  30 uL
  • 95 C 1min (95 C 10s, 65C 15s, 72 C 45s)x8

Second round PCR

  • Pause PCR program and add following to each
1st round PCR mix 30 uL
KAPA Robust mix 35 uL
Adapter 1 2 uL
Adapter 2 Barcode 2 uL
SYBR Green 1 .56 uL
water 30.44 uL
----------------------
Total 100 uL

1 min 95C, (95 C 10s, 65C 15s, 72 C 45s)x3, (95 C 10s, 72C 1 min)x4, 72C 3min



Bead purfication

  • Add 100 uL beads to each, mix, and let sit at RT for 8min
  • Transfer tubes to magnetic rack and let sit for 5 min
  • Remove supernatant
  • 2x 80% EtOH wash
  • Sit for 15 min
  • Elute in 23 uL Qiagen buffer


Results

  • There is a 1 cycle difference between PC and EtOH, implicating 1/2 the DNA is lost
  • Even though the buffer for the Pol I reactions is the same as tagmentation buffer, tagmentation is very inefficient. ALS is prepared as diluted KOH only (no DTT or EDTA). Not sure what is happening. Only differences are K and CL ions from ALS/NS, and dNTP's/N6 primer from Pol I mix
  • When the Pol I reaction was run, it is entirely inhibited meaning the enzymes are somehow competing (even though Pol I is killed)
  • In any case, EtOH continues to be the best option, and I will only use this in the future


File:11-14-12 pcr.jpg File:11-14-12 gel1.jpg